Lettuce Plant Resistant to Downy Mildew and Resistance Gene

ABSTRACT

Provided herein is a lettuce plant that is resistant to downy mildew, more specifically a lettuce plant that includes a mutated gene that confers broad spectrum resistance to oomycetes in lettuce, more specifically Bremia lactucae. Furthermore also provided herein are a resistance gene and a method for obtaining a lettuce plant that is resistant to downy mildew, wherein the method includes the step of mutating a gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States national phase of InternationalApplication No. PCT/EP2019/083651 filed Dec. 4, 2019, and claimspriority to International Application No. PCT/EP2018/085244 filed Dec.17, 2018, the disclosures of which are hereby incorporated by referencein their entirety.

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety. The name of the text filecontaining the Sequence Listing is 2102739_ST25.txt. The size of thetext file is 21,911 bytes, and the text file was created on May 19,2021.

DESCRIPTION

The present invention relates to a lettuce plant that is resistant todowny mildew, more specifically to a lettuce plant that comprises amutated gene that confers broad spectrum resistance to Bremia lactucaein lettuce. Furthermore the present invention relates a resistance geneand a method for obtaining a lettuce plant that is resistant to downymildew, wherein the method comprises the step of mutating a gene.

Downy mildew refers to several types of oomycete microbes that areparasites of plants. Downy mildew can originate from various species,but mainly of Peronospora, Plasmopara and Bremia. Downy mildew is aproblem in many food crops, in for example in lettuce caused by Bremialactucae, affecting the production of this crop worldwide. Plants thatare being affected include food crops such as brassicas (e.g. cabbage),potatoes, grape, spinach, lettuce, onion, tomato, cucumber plants. Downymildew infection show symptoms of discoloured areas on upper leafsurfaces in combination with white, grey or purple mould located on theother side of the leaf surface below. Disease is spread from plant toplant by airborne spores.

Lettuce, mostly known as Lactuca sativa, but also including Lactucaspecies such as L. serriola, L. saligna or L. virosa, is a veryimportant crop worldwide. Some of the most popular varieties availableare Iceberg, Romaine, Butterhead, Batavia and Oakleaf. There are manyplant pathogens that affect L. sativa, and some of the diseases causedby these pathogens are downy mildew, Sclerotinia rot, powdery mildew,fusarium wilt of which the most important disease is lettuce downymildew, which is caused by the B. lactucae, an oomycete pathogen thatbelong to Peronosporaceae.

For some vegetable crops, such as lettuce, cultivars with resistance todowny mildew are available. However, the pathogen under pressure willmutate to break down the disease resistance and new disease resistancein crops is needed to control infection. Especially in lettuce theoccurrence of resistant downy mildew is particularly complex as thereare many different races, and new resistant downy mildew speciesemerging all the time.

In lettuce, infection of B. lactucae result in yellow to pale greenlesions that eventually become necrotic due to secondary pathogensleading to major crop losses. Fungicides can be used to control B.lactucae, but eventually B. lactucae becomes immune to these chemicals,because over time the pathogen also acquires resistance to fungicides.Furthermore, there are multiple lettuce varieties available that areresistant to B. lactucae but resistance is quickly overcome because newBremia races develop rapidly. Therefore, it is of the utmost importanceto find other methods to control B. lactucae infection. Most preferablyis to identify a resistance gene that gives broad resistance against B.lactucae and to provide for lettuce plants that are resistant to downymildew. Therefore, identification of resistance genes is a promisingalternative.

Considering the above, there is a need in the art for to provide plantsthat are resistant to downy mildew and wherein plants have a broadspectrum resistance against this pathogen. Furthermore, it is an objectof present invention to provide a method to obtain such downy mildewresistant plants.

SUMMARY

It is an object of the present invention, amongst other objects, toaddress the above need in the art. The object of present invention,amongst other objects, is met by the present invention as outlined inthe appended claims.

Specifically, the above object, amongst other objects, is met, accordingto a first aspect, by the present invention by a lettuce plant that isresistant to downy mildew, wherein said plant comprises one or moremutations in a SL7 gene, wherein said SL7 gene encodes for a proteinsequence of SEQ ID No. 2 or having at least 90% sequence identity withSEQ ID No. 2, preferably at least 95%, more preferably at least 98%,even more preferably at least 99%, most preferably 100%, wherein the oneor more mutations in the SL7 gene result in amino acid substitutions onposition 38 in the SL7 protein represented by SEQ ID No. 2. Preferablythe serine (S) at position 38 in SEQ ID No. 2 is mutated to asparagine(N). The mutated SL7 gene is a dominant resistance gene, and may behomozygous or heterozygous present in a downy mildew resistant lettuceplant.

The majority of disease resistance genes in plants encodenucleotide-binding site leucine-rich repeat proteins, also known asNBS-LRR proteins (encoded by R genes). These proteins are characterizedby nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domainsas well as variable amino- and carboxy-terminal domains and are involvedin the detection of diverse pathogens, including bacteria, viruses,fungi, nematodes, insects and oomycetes. There are two major subfamiliesof plant NBS-LRR proteins defined by the Toll/interleukin-1 receptor(TIR) or the coiled-coil (CC) motifs in the amino-terminal domain andare both involved in pathogen recognition.

A leucine-rich repeat (LRR) is a protein structural domain composed ofrepeating 20 to 30 amino acid stretches that forms an a/I3 horseshoefold. The domain is rich in the hydrophobic amino acid leucine. Theregion between the helices and sheets is the protein's hydrophobic coreand is tightly sterically packed with leucine residues. On averageclassical NBS-LLR genes comprise six LRR regions. The SL7 gene is not aclassical NBS-LRR gene, since the SL7 protein does not comprise multipleLRR regions, and it does not comprise the NBS domain. The SL7 genecontains only one LRR region, in which the SL7 gene differs from othercases of R genes where multiple LRR regions and the NBS domain arepresent. It is thought that those domains determine effector recognitionand therefore disease susceptibility/resistance. The presence of the SL7resistance gene will decrease the chances of the pathogen overcoming theresistance, as often seen with the R genes. Even so, combined with Rgenes, disease resistance (e.g. against downy mildew) may even befurther improved.

For the first time a resistance gene has been found in a lettuce plantthat is located on chromosome 3 and that can be linked to plant diseaseresistance. This SL7 gene of present invention gives resistance toBremia lactucae races Bl17 to Bl35, with the exception of Bl16, Bl20,Bl21, Bl23, and Bl27. Preferably, spectrum resistance to Bremia lactucaein lettuce comprises resistance to Bremia lactucae of at least racesBl17, Bl18, Bl22, Bl24 to Bl26, and Bl28 to Bl35.

To demonstrate that the SL7 gene is related to Bremia resistance, thisputative resistance gene has been silenced by tobacco rattle virus(TRV)-based virus-induced gene silencing (VIGS) to induce susceptibilityto B. lactucae infection in resistant L. saligna lettuce linescontaining the SL7 resistance gene. With VIGS it was demonstrated thatthe SL7 gene was associated with downy mildew resistance, VIGS genesilencing was used to create Bremia-susceptibility in resistant Lactucaspecies. Resistant lettuce plants were transient transformed with an SL7silencing construct and made susceptible to B. lactucae infection, thusby removing the SL7 gene via virus induced gene silencing. According toanother preferred embodiment, the present invention relates to theLettuce plant, wherein the one or more mutations in the SL7 gene resultin amino acid substitutions in the region comprised of amino acidpositions 6 to 147 in the SL7 protein represented by SEQ ID No.2. Theregion comprised of amino acids 6 to 147 is an LRR region of SL7,comprising at least one or more LLR domains, preferably at least two,more preferably at least three, most preferably four LLR domains.

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 11 inthe SL7 protein represented by SEQ ID No.2. Preferably the threonine (T)at position 11 in SEQ ID No. 2 is mutated to arginine (R).

According to another preferred embodiment, the present invention relatesto the Lettuce plant, wherein the one or more mutations in the SL7 generesult in amino acid substitutions in the region comprised of amino acidpositions 26 to 72 in the SL7 protein represented by SEQ ID No.2. Theregion comprised of amino acids 26 to 72 is a single LRR domain of SL7.

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 40 inthe SL7 protein represented by SEQ ID No.2. Preferably the lysine (K) atposition 40 in SEQ ID No. 2 is mutated to threonine (T).

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 48 inthe SL7 protein represented by SEQ ID No.2. Preferably the serine (S) atposition 48 in SEQ ID No. 2 is mutated to asparagine (N).

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 61 inthe SL7 protein represented by SEQ ID No.2. Preferably the threonine (T)at position 61 in SEQ ID No. 2 is mutated to serine (S).

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 69 inthe SL7 protein represented by SEQ ID No.2. Preferably the isoleucine(I) at position 69 in SEQ ID No. 2 is mutated to valine (V).

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 84 inthe SL7 protein represented by SEQ ID No.2. Preferably the cysteine (C)at position 84 in SEQ ID No. 2 is mutated to arginine (R).

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 91 inthe SL7 protein represented by SEQ ID No.2. Preferably the aspartic acid(D) at position 91 in SEQ ID No. 2 is mutated to asparagine (N).

According to yet another preferred embodiment, the present inventionrelates to the Lettuce plant, wherein the one or more mutations in theSL7 gene further result in amino acid substitutions on position 129 inthe SL7 protein represented by SEQ ID No.2. Preferably the valine (V) atposition 129 in SEQ ID No. 2 is mutated to isoleucine (I).

According to another preferred embodiment, the present invention relatesto the Lettuce plant, wherein the mutations in the SL7 gene result inamino acid substitutions at position 11, 38, 40, 48, 61, 69, 84, 91 and129 in the SL7 protein represented by SEQ ID No.2. Preferably themutations are T11R, S38N, K40T, S48N, T61S, I69V, C84R, D91N, and V129Irespectively. An SL7 resistance gene of present invention that encodesfor the protein that comprises all the above mutations is represented bySEQ ID No. 3 and encodes for the SL7 protein is represented by SEQ IDNo. 4.

According to another preferred embodiment, the present invention relatesto the Lettuce plant, wherein the mutations in the SL7 gene furtherresult in amino acid substitutions at position 11, 40, and 84 in the SL7protein represented by SEQ ID No.2. Preferably the mutations are T11R,S38N, K40T, and C84R.

According to another preferred embodiment, the present invention relatesto the Lettuce plant, wherein the SL7 gene that comprises one or moremutations and encodes for the protein sequence represented by SEQ ID No.4. The mutated SL7 gene, being the SL7R gene, is represented by SEQ IDNo. 3. Sequencing experiments showed that the protein encoded by theSL7R gene from the resistant plant compared with the protein encoded bythe SL7 gene of a plant that is susceptible differs in several aminoacids that have been mutated. In particular mutations in the LRR regionof the SL7 protein in the amino acid 6 to 147 are of importance toprovide resistance to downy mildew, more specifically in the LLR domainof the SL7 protein in the amino acid region of 26 to 72. The downymildew is caused in the Lettuce plant by an oomycete, more preferablyBremia lactucae.

According to yet another preferred embodiment, the present inventionrelates to the lettuce plant, wherein the plant is selected from Lactucasativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactucaaculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactucaviminea, preferably Lactuca sativa.

According to a preferred embodiment, the present invention relates tothe lettuce plant, wherein the mutations in the SL7 gene are obtainableby gene editing techniques, preferably by mutagenesis (e.g. EMS) and/orCRISPR/Cas.

According to another preferred embodiment, the present invention relatesto the lettuce plant, wherein the lettuce plant is resistant to downymildew caused by one or more of Bremia lactucae selected from the groupof race Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35. A lettuce plant ofpresent invention comprising the SL7 resistance gene is susceptible todowny mildew caused by Bremia lactucae Bl16, Bl20, Bl21, Bl23, and Bl27.Preferably, spectrum resistance to Bremia lactucae in the lettuce ofpresent invention comprises resistance to Bremia lactucae of at leastraces Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35.

According to yet another preferred embodiment, the present inventionrelates to the lettuce plant, wherein the resistance gene SL7R of SEQ IDNo. 3 is obtainable from deposit number NCIMB 42785.

The present invention, according to a second aspect, relates to seedsproduced by the lettuce plant of present invention. The seed comprisesthe SL7R gene as described above.

The present invention, according to a third aspect, relates to aresistance gene SL7R that confers resistance to Bremia lactucae inlettuce plants, wherein the gene comprises a coding sequence of SEQ IDNo. 3 or having at least 90% sequence identity with SEQ ID No. 3,preferably at least 95%, more preferably at least 98%, most preferablyat least 99%, most preferably 100%. The SL7R gene is a dominant gene.SEQ ID No.3 represents the coding nucleotide sequence of SL7R gene ofLactuca saligna and encodes for the SL7R protein sequence represented bySEQ ID No.4. SEQ ID No.4 represents the SL7R protein sequence of Lactucasaligna and lettuce plants that express this protein show completeresistance to downy mildew.

According to a preferred embodiment, the present invention relates toresistance gene SL7R, wherein the gene encodes for a SL7R protein thathas at least 85% sequence identity with SEQ ID No. 4, preferably atleast 90%, more preferably at least 95%, most preferably at least 98%,most preferably 100%.

According to another preferred embodiment, the present invention relatesto the resistance gene SL7R, wherein resistance to Bremia lactucae inlettuce comprises resistance to Bremia lactucae of race Bl17, Bl18,Bl22, Bl24 to Bl26, Bl28 to Bl35. Preferably, spectrum resistance toBremia lactucae in lettuce comprises resistance to Bremia lactucae of atleast races Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35.

According to yet another preferred embodiment, the present inventionrelates to the resistance gene SL7R, wherein the plant is selected fromLactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola,Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica,Lactuca viminea, preferably Lactuca sativa.

The present invention, according to a further aspect, relates to amethod for obtaining a lettuce plant that is resistant to downy mildew,wherein the method comprises the steps of,

-   -   a) crossing a lettuce plant comprised of the resistance gene        SL7R of present invention with a lettuce plant that does not        comprise said SL7R gene,    -   b) optionally, selfing the plant obtained in step a) for at        least one time,    -   c) selecting the plants that are resistant to downy mildew.        In the method of present invention the lettuce plant is selected        from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca        serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis,        Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.

The present invention, according to a further aspect, relates to amethod for obtaining a lettuce plant that is resistant to downy mildew,wherein the method comprises the step of providing one or more mutationsin a SL7 gene of a lettuce plant, resulting in a SL7R resistance gene ofpresent invention. The SL7 gene comprises a coding sequence that has atleast 90% sequence identity with SEQ ID No. 1, preferably at least 95%,more preferably at least 98%, most preferably at least 99%, mostpreferably 100%. SEQ ID No.1 represents the coding nucleotide sequenceof the SL7 gene of Lactuca sativa. This sequence is the wild typesequence and does not contain the mutations as compared to theresistance gene of present invention.

According to another preferred embodiment, the present invention relatesto the method, wherein the one or more mutations in the SL7 gene resultin amino acid substitutions in the region comprised of amino acidpositions 6 to 147, preferably comprised of amino acid positions 26 to72, in the SL7 protein represented by SEQ ID No.2. Mutations are locatedin the LLR region of amino acid positions 6 to 147, preferably in thesingle LRR domain that is located from amino acid 26 to 72 of the SL7protein.

According to yet another preferred embodiment, the present inventionrelates to the method, wherein the one or more mutations in the SL7 genecomprise amino acid substitutions at position 11, 38, 40, and 84 in theSL7 protein represented by SEQ ID No.2.

According to yet another preferred embodiment, the present inventionrelates to the method, wherein the one or more mutations in the SL7 genefurther comprises amino acid substitutions at position 48, 61, 69, 91and 129 in the SL7 protein represented by SEQ ID No.2.

According to a preferred embodiment, the present invention relates tothe method, wherein the SL7 gene that comprises one or more mutations,being the SL7R gene, is represented by SEQ ID No. 3 and encodes for theprotein sequence represented by SEQ ID No. 4. SEQ ID No.4 represents theSL7R protein sequence of Lactuca saligna. Lettuce, such as L. sativathat express the protein of SEQ ID No.4 is resistant to downy mildew.

According to a preferred embodiment, the present invention relates tothe method, wherein the mutations in the SL7 gene are obtained by geneediting techniques, preferably by mutagenesis and/or CRISPR/Cas. Thelettuce plant comprising the mutations in the SL7 gene is selected fromLactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola,Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica,Lactuca viminea, preferably Lactuca sativa. A lettuce plant comprised ofthe mutations in the SL7 gene gives a high downy mildew resistancephenotype. A plant having this resistant phenotype can be obtained viause of gene editing and/or mutation techniques, such as EMS mutagenesisor CRISPR/Cas in concert with cloning techniques on the SL7 gene togenerate disease resistant crops. Mutations induced by gene editingtechniques such as mutagenesis, CRISPR/Cas, transgenic techniques, orothers can be regarded as non-natural mutations. Alternatively, a SL7Rgene can be brought into the plant by means of transgenic techniques orby introgression.

The present invention, according to a further aspect, relates to the useof a gene construct for introducing a resistance gene into the genome ofa plant or plant cell, wherein the gene construct is comprised of theresistance gene SL7R of present invention which is operably linked toexpression providing sequences in said plant. The resistance gene ofpresent invention may be transferred (e.g. by transformation ortransfection) into plants, such as lettuce plants, using a plasmid ofvector or linear gene construct that comprises the SL7R resistance geneof present invention or wherein the gene comprises a coding sequencethat has at least 90% sequence identity with SEQ ID No. 3. Theresistance gene SL7R encodes for a SL7R protein that has at least 85%sequence identity with SEQ ID No. 4. The Resistance gene SL7R, afterbeing transferred into the lettuce plant would provide resistance toBremia lactucae i.e. resistance to Bremia lactucae of race Bl17, Bl18,Bl22, Bl24 to Bl26, Bl28 to Bl35.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further detailed in the following examplesand figures wherein:

FIG. 1: shows the number (#) of leaves of Lettuce (Y-axis) that areresistant or susceptible to Bremia lactucae after VIGS silencing ofeither SL7R (by 1A and 1B), or gene Lsa042767 (by 2A or 2B) (X-axis).The SL7R gene has been silenced in these plants using VIGS genesilencing and subsequently infected with Bremia lactucae (Bl30). On thex-axis from left to right: sample leaves of plants in which the SL7Rgene is silenced using silencing construct 1A or 1B, sample leaves ofplants in which gene Lsa042767 is silenced using silencing construct 2Aor 2B, sample leaves of plants in which PDS is silenced, sample leavesof plants of susceptible parent R273. In the samples with a resistantphenotype, there is no Bremia present. In the samples with susceptiblephenotypes, Bremia is present. As expected with transient genesilencing, VIGS gene silencing does not result in fully 100% silencingof the SL7R gene in all plants. However, the leaves from plants whereinthe resistance gene has been silenced by VIGS silencing, showed a highernumber of susceptible leaves when infected with Bremia as compared toplants where the SL7R gene was not silenced. Indeed no susceptibleleaves were observed when SL7R expression was not affected by VIGS.

FIG. 2: shows quantification of Bremia actin in Lettuce after VIGS genesilencing. In case SL7R gene expression levels were VIGS silenced inlettuce infected with Bremia (Bl30), expression levels of Bremia actinincreased dramatically. The Bremia expression levels in the leaves ofplants that showed to be resistant or susceptible to downy mildew aftergene silencing, were collected and RNA was isolated to determine theexpression levels of Bremia by qPCR. The transcription levels of Bremialactuca was determined by the transcripts of a Bremia house keeping gene(actin) in relation to the lettuce house keeping gene TUA-3 in a set ofleave samples of Lettuce plants of the experiment of FIG. 1. Leaves ofthe plant that were susceptible to Bremia lactucae, showed hightranscriptional levels of the Bremia lactucae house keeping gene actin,indicating the susceptibility corresponds with low SL7R gene expressiondue to VIGS silencing.

FIG. 3: shows SL7R expression levels in SL7R VIGS silenced lettuce linesinfected with Bremia (Bl30), determined by qPCR. The transcriptionlevels of Bremia lactuca was determined by the transcripts of a Bremiahouse keeping gene (actin) in relation to the lettuce house keeping geneTUA-3 of Bremia lactucae were determined in leave samples of L. sativaplants of the experiment of FIG. 1. Leaves of the plants that wereresistant to Bremia lactucae showed to have a high SL7R gene expressionand low transcriptional levels of the Bremia lactucae house keepinggene. Leaves of the plant that were susceptible to Bremia lactucae,showed low SL7R gene expression (because of VIGS silencing the gene) andhigh transcriptional levels of the Bremia lactucae house keeping gene,indicating the susceptibility corresponds with low SL7R gene expression.

FIG. 4: shows an overview of the disease test performed with the mostrecent isolates of Bremia Bl16 to Bl35 on L. sativa lines Cobham GreenR273, Green Towers, Vanity and SL7R. SL7R is a lettuce plant (L. sativa)of present invention comprising the SL7R resistance gene. The plant ofpresent invention shows to be resistant to most downy mildew isolates,with the exception of Bremia lactucae Bl16, Bl20, Bl21, Bl23, and Bl27.

FIG. 5: shows the alignment of the amino acid sequence region of 1 to150 amino acids, including the LLR region (amino acid 6 to 147) and thesingle LRR domain (amino acid 26 to 72) of SL7 (SEQ ID No.2) and theSL7R (SEQ ID No.4) protein. The mutations between the two proteinsequences have been indicated in the boxed areas. The SL7R proteincomprises amino acid substitutions at position 11 (T→R), 38 (S→N), 40(K→T), 48 (S→N), 61 (T→S), 69 (I→V), 84 (C→R), 91 (D→N) and 129 (V→I).

FIG. 6: shows the cDNA sequence (SEQ ID No. 1) encoded by the SL7 geneof Lactuca sativa.

FIG. 7: shows the protein sequence (SEQ ID No. 2) encoded by the SL7gene of Lactuca sativa.

FIG. 8: shows the cDNA sequence (SEQ ID No. 3) encoded by the SL7R geneof Lactuca saligna.

FIG. 9: shows the protein sequence (SEQ ID No. 4) encoded by the SL7Rgene of Lactuca saligna.

DETAILED DESCRIPTION Examples

Gene Mapping SL7 Resistance Gene in L. saligna

Gene mapping experiments were done to identify the of the Bremia (Bremialactucae) resistance gene SL7R from Lactuca saligna. SL7R was originallyisolated from L. saligna lettuce accession LAC0364. A dominantresistance gene was mapped on chromosome 3, Lettuce genome V8. The SL7Rresistance gene is the first Bremia resistance gene described and mappedon chromosome 3 in Lettuce.

After fine mapping 12000 plants two putative genes were present. TheSL7R region is currently flanked by two markers based on a SNP atposition V8_3_200695773 and a SNP at position V8_3_200770104. The twocandidate genes are designates as Lsa011563 and Lsa042767. VIGSsilencing was used to silence both genes independently in a resistantsource (L. saligna), see below. These experiments indicated that whenresistance gene Lsa011563 was silenced the plants became susceptibleafter Bremia infection, whereas when gene Lsa042767 was silenced, theplants remained resistant. This confirms that the Lsa011563 gene providethe plant resistance against Bremia. This resistance gene is renamed tothe resistance gene SL7R of present invention.

Construction of SL7 Construct and Transformation into Lettuce (L.saligna).

After gene mapping two candidate resistance genes were found anddesignated as Lsa011563 (=SL7R) and Lsa042767. To identify which gene(or both) are responsible for the observed resistance, VIGS silencingcan be used to silence both genes independently in the resistant sourceL. saligna. Therefore, two VIGS-constructs were made per gene, forLsa011563 (=SL7) (1A and 1B) and for Lsa042767 (2A and 2B) and cloned inthe K20 vector (See Table 1 for sequences, respectively SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8). Thus two SL7R specific VIGSconstructs were made (1A and 1B) and two construct that target adifferent gene Lsa042767 (2A, 2B) can be used as a negative control. Themultiple constructs of above were transformed into lettuce (L. sativa)using co-cultivation with agrobacterium (GV3101) to study the SL7Rfunction. The two resistance candidate genes are individual silenced inVIGS independent experiments. With the leaves of VIGS-experimentsindependent disease tests (see below) were performed to observe thatwhen SL7R was silenced, plants became susceptible to Bremia.

TABLE 1 VIGS-constructs Sequence Lsa011563_1ATTCATGTAGCTTCTTCAGTCACCATGTTGGAAATAGGTAATATT (SEQ ID No. 5)TCAGGGCTTAATGATGAACTGTGGAGAAGTGCTTTCAAGTATCTTGGGAAACTTGAAAAGTTATACATTCGTGGGTGTAATGAAATAAGATATTTGTGGCAATCAGAAGTAGAGGCAAGTAAGTCTCTAGTGAATTTAAGGAATTTGGATGTGAGTGATTGTTCAAATCTGGTGGGTTTAGGAGAGAAAGTGGAGGATAACTCTGGAAGCATCCAGACGTTTATTAGGATGTTGTCTATAGCACGTTGTGAGA Lsa011563_1BTGAGTTACCTTGGAATAGGAGGATTGAAGAAGCCCATCTCAGAG (SEQ ID No. 6)TGGGGCCCACAGAATTTCCCAACCTCACTCGAGCACTTAATGTTAAATGGCGGAATATATGATGATGTGAAAAACTTTGATCAATTGTCGCATCTTTTTCCTTCATCTCTTGCTTCTCTTTCGATAACGGGATTTCAGAAACTTGAATCAGTTTCATTGGGACTCCAAAACCTCACCTTTCTCCAGCGTCTCTCTGTTTCCAAGTGCCCAAAGATGTTACATCTACCAGAAAAGTTGCTTCCTTCGCTTTTGTCTTTGAG Lsa042767_2AGTGGAGATCAAGCTGGATTATAAGAAGGATTTGTTTGATGGGAA  (SEQ ID No. 7)GAGGAATATTGTCACGGCGGAGGAGATAGAGAGCGGGATAAGGC GGCTGATGGAGGATGACGATGTAAGAGAAAAGATAAAAGAGATGGGGAAAAAGAGCAAAGCGACTGTTAAAGAGGGAGGTTCGTCTTA CGCTTCT Lsa042767_2BCACATTCTTGGAATTAGAAACACGCCCAATCGAGTCGTTGTCTA  (SEQ ID No. 8)CCGACAGCAGGATACCGTCTGTGTATCCGGTAGGACCTGTACTG AACCTAGAAGACGGTGCCGGAACACCGCCGGAAAGTGACGTCATCAGCTGGTTGGACAATCAACCACCTTCCTCGGTTGTTTTCTTGTGTTTTGGGAGTCTGGGATGTTTTGATGAAGTCCAAGTGAAGGAGATTGCATATGCTTTAGAGCGAAGCGGGCGTTCTTTCTTGTGGTC ACTAC

SL7R Gene Silencing Experiment Using Virus Induced Gene Silencing (VIGS)

Tobacco rattle virus (TRV)-derived VIGS vectors have been abundantlydescribed to study gene function in Arabidopsis thaliana, Nicotianabenthamiana, Solanum esculentum and other plants (see for example HuangC, Qian Y, Li Z, Zhou X.: Virus-induced gene silencing and itsapplication in plant functional genomics. Sci China Life Sci. 2012;55(2):99-108).

Briefly, Lettuce containing SL7 were silenced for SL7R by VIGS.Independent of SL7R silencing the PDS gene is silenced as well thatserves as positive control to indicate if VIGS is working and todetermine the efficiency. PDS is involved in carotenoid biosynthesis andis the first step in lycopene biosynthesis. This step is catalyzed byphytoene desaturase (PDS). When silencing of the PDS gene is achieved,this results in bleached leaves.

Furthermore, all plants that were SL7R-VIGS inoculated were harvestedand put in a tray and sprayed with Bremia to test the effect of the genesilencing on disease resistance.

Disease Test and Biotest for Downy Mildew in Lettuce

Leaves of resistant plants transiently transformed with the abovedescribed VIGS constructs (1A, 1B, 2A, 2B and PDS), were put in trayswith moistened paperboard and infected with Bremia race 30. The infectedseedlings are suspended in 20 mL water, filtered by cheesecloth and theflow-through is collected in a spray flask. One tray is spray-inoculatedwith the Bremia lactucae suspension. The trays are covered with a glassplate and stored in a climate chamber at 15° C. (12 hours of light). Ablack, opaque foil is placed over the trays for one day to improvegrowth of B. lactucae. After one day, the foil is removed. Experimentswere performed in triple, and eight to ten days after infection leavesare phenotypically scored by eye on the presence of Bremia, i.e. beingsusceptible or resistant (FIG. 1).

Disease resistance tests show that resistance gene SL7R providesresistance to most Bremia races from Bl:15 to Bl:35, with the exceptionsbeing Bl:16, BL20, Bl21, Bl23, and Bl:27. Furthermore a qPCR wasperformed to determine SL7R expression.

A single gene line comprising the SL7R gene used internally to testBremia diagnostic samples is R290. Seeds of this line are deposited atNCIMB (NCIMB Limited, Ferguson Building; Craibstone Estate, BucksburnABERDEEN, Scotland, AB21 9YA United Kingdom) on 12 Jul. 2017 under thenumber NCIMB 42785.

Determine Bremia Expression in Lettuce Comprising the SL7R Gene

A number of gene expression experiments were conducted in lettucetissues obtained from the VIGS experiment as outlined above, todetermine SL7R expression. The response of lettuce leaves to Bremialactucae infection was examined and gene expression studies were used toassess VIGS analysis.

To obtain more insight in the response of lettuce to infection withBremia, leaves of resistant and susceptible plants were harvested. cDNAwas synthesized from RNA that had been isolated from infected leaves.The expression of SL7R was assessed in lettuce by conducting qPCR.Expression of Bremia lactucae actin and expression of SL7R were analyzedby qPCR using the primers as set out in Table 2. (SEQ ID No.9, SEQ IDNo.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, and SEQ ID No.14,respectively).

TABLE 2 Primer name Sequence SL7QPCR-F 5′-TCCAAGTATTGATGCCTCCTT-3′(SEQ ID No. 9) SL7QPCR-R 5′-CACTCTGAGATGGGCTTCTTC-3′ SEQ ID No. 10)B. lactucae 5′-GCGAGAAATTGTGCGTGATA-3′ actin Fwd (SEQ ID No. 11B. lactucae 5′-ACTCGGCTGCAGTCTTCATT-3′ actin Rv (SEQ ID No. 12) LsTUA-3F5′-CTTCTTAGTGTTCAATGCTGTTGG-3′ (SEQ ID No. 13) LsTUA-3R5′-GAAGGGTAGATAGTGAAACCGAGC-3′ (SEQ ID No. 14)

FIG. 2 shows the results of a qPCR of housekeeping gene Bremia actin.Values on the y-axis are CT values (the fold increase is calculated as2{circumflex over ( )}−(Ct Bremia actin−Ct TUA3A). On the x-axis fromleft to right: sample leaf of a plant in which PDS is silenced, sampleleaves of plants of susceptible parent R273, sample leaves of plants inwhich the SL7R gene is silenced using silencing construct 1B, sampleleaves of plants in which the SL7R gene is silenced using silencingconstruct 2B. In the samples with a resistant (R) phenotype, there is noor almost no Bremia present. In the samples with susceptible (S)phenotypes, high transcription levels of the housekeeping gene Bremiaactin were measured.

In addition, FIG. 3 shows that in leaves of the plants that areresistant to Bremia, little to no Bremia was detected and that the levelof SL7R expression was very high. The leaves that originate from plantsthat are susceptible to Bremia, showed the opposite pattern, a highlevel of Bremia and low levels of SL7R expression.

1. A lettuce plant that is resistant to downy mildew, comprising one ormore mutations in a SL7 gene, wherein said SL7 gene encodes for aprotein having the sequence of SEQ ID No. 2 or having at least 90%sequence identity with SEQ ID No. 2, wherein the one or more mutationsin the SL7 gene result in an amino acid substitution at position 38 inthe SL7 protein represented by SEQ ID No.2.
 2. The lettuce plantaccording to claim 1, wherein the one or more mutations in the SL7 generesult in amino acid substitutions in the region comprising amino acidpositions 6 to 142 in the SL7 protein represented by SEQ ID No.2.
 3. Thelettuce plant according to claim 1, wherein the one or more mutations inthe SL7 gene further result in an amino acid substitution at position 11in the SL7 protein represented by SEQ ID No.2.
 4. The lettuce plantaccording to claim 1, wherein the one or more mutations in the SL7 genefurther result in an amino acid substitution at position 40 in the SL7protein represented by SEQ ID No.2, preferably K40T.
 5. The lettuceplant according to claim 1, wherein the one or more mutations in the SL7gene additionally result in an amino acid substitution at position 48 inthe SL7 protein represented by SEQ ID No.2.
 6. The lettuce plantaccording to claim 1, wherein the one or more mutations in the SL7 geneadditionally result in an amino acid substitution at position 61 in theSL7 protein represented by SEQ ID No.2, preferably T61S.
 7. The lettuceplant according to claim 1, wherein the one or more mutations in the SL7gene additionally result in an amino acid substitution at position 69 inthe SL7 protein represented by SEQ ID No.2.
 8. The lettuce plantaccording to claim 1, wherein the one or more mutations in the SL7 geneadditionally result in an amino acid substitution at position 84 in theSL7 protein represented by SEQ ID No.2.
 9. The lettuce plant accordingto claim 1, wherein the one or more mutations in the SL7 geneadditionally result in an amino acid substitution at position 91 in theSL7 protein represented by SEQ ID No.2.
 10. The lettuce plant accordingto claim 1, wherein the one or more mutations in the SL7 geneadditionally result in an amino acid substitution at position 129 in theSL7 protein represented by SEQ ID No.2.
 11. The lettuce plant accordingto claim 1, wherein the one or more mutations in the SL7 gene furtherresult in amino acid substitutions at position 11, 40, and 84 in the SL7protein represented by SEQ ID No.2.
 12. The lettuce plant according toclaim 1, wherein the one or more mutations in the SL7 gene result inamino acid substitutions at positions 11, 38, 40, 48, 61, 69, 84, 91 and129 in the SL7 protein represented by SEQ ID No.2.
 13. The lettuce plantaccording to claim 1, wherein the SL7 gene that comprises one or moremutations encodes for a protein having the sequence of SEQ ID No.
 4. 14.The lettuce plant according to claim 1, wherein the lettuce plant isselected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactucaserriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactucatatarica, and Lactuca viminea.
 15. The lettuce plant according to claim1, wherein downy mildew is caused by Bremia lactucae.
 16. The lettuceplant according to claim 1, wherein the lettuce plant is resistant todowny mildew caused by one or more of Bremia lactucae selected fromraces Bl17, Bl18, Bl22, Bl24 to Bl26, Bl28 to Bl35.
 17. The lettuceplant according to claim 1, wherein the plant comprises an SL7R genehaving the nucleotide sequence of SEQ ID No.
 3. 18. Seed produced by alettuce plant according to claim 1, wherein the seed comprises the SL7gene.
 19. (canceled)
 20. (canceled)
 21. (canceled)
 22. (canceled)
 23. Amethod for obtaining a lettuce plant that is resistant to downy mildew,comprising the steps of, a) crossing a first lettuce plant comprising aresistance gene SL7R having the nucleotide sequence of SEQ ID No. 3 witha second lettuce plant that does not comprise said SL7R gene, therebyproducing a first offspring plant b) optionally, selfing the offspringplant obtained in step a) for at least one time, thereby producing asecond offspring plant c) selecting one or more first and/or secondoffspring plants that are resistant to downy mildew.
 24. (canceled) 25.(canceled)
 26. (canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)30. (canceled)
 31. The method according to claim 23, wherein the firstand/or second lettuce plant is selected from Lactuca sativa, Lactucavirosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactucageorgica, Lactuca perennis, Lactuca tatarica, and Lactuca viminea. 32.(canceled)